Rb and N-Ras Function Together to Control Differentiation in the Mouse

Rb and N-ras Function Together to Control Differentiation in the Mouse

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Citation Takahashi, C., R. T. Bronson, M. Socolovsky, B. Contreras, K. Y. Lee, T. Jacks, M. Noda, R. Kucherlapati, and M. E. Ewen. 2003. “Rb and N-Ras Function Together To Control Differentiation in the Mouse.” Molecular and Cellular Biology 23 (15): 5256–68. doi:10.1128/ MCB.23.15.5256-5268.2003.

Citable link http://nrs.harvard.edu/urn-3:HUL.InstRepos:41543053

Terms of Use This article was downloaded from Harvard University’s DASH repository, and is made available under the terms and conditions applicable to Other Posted Material, as set forth at http:// nrs.harvard.edu/urn-3:HUL.InstRepos:dash.current.terms-of- use#LAA MOLECULAR AND CELLULAR BIOLOGY, Aug. 2003, p. 5256–5268 Vol. 23, No. 15 0270-7306/03/$08.00ϩ0 DOI: 10.1128/MCB.23.15.5256–5268.2003 Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Rb and N-ras Function Together To Control Differentiation in the Mouse Chiaki Takahashi,1† Roderick T. Bronson,2,3 Merav Socolovsky,4 Bernardo Contreras,1 Kwang Youl Lee,1‡ Tyler Jacks,5 Makoto Noda,6 Raju Kucherlapati,7 and Mark E. Ewen1* Department of Medical Oncology and Medicine, Dana-Farber Cancer Institute and Harvard Medical School,1 and Rodent Histopathology Core3 and Harvard-Partners Center for Genetics and Genomics,7 Harvard Medical School, Boston, Massachusetts 02115; Department of Pathology, Tufts University Schools of Medicine and Veterinary Medicine, 2

Boston, Massachusetts 02111 ; Whitehead Institute for Biomedical Research, Massachusetts Institute of Downloaded from Technology, Cambridge, Massachusetts 021424; Department of Biology and Howard Hughes Medical Institute, Center for Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts 021395; and Department of Molecular Oncology, Kyoto University Graduate School of Medicine, Sakyo-ku, Kyoto 606-8501, Japan6

Received 8 November 2002/Returned for modiﬁcation 7 January 2003/Accepted 30 April 2003

The product of the retinoblastoma tumor suppressor gene (Rb) can control cell proliferation and promote dif-

ferentiation. Murine embryos nullizygous for Rb die midgestation with defects in cell cycle regulation, control http://mcb.asm.org/ of apoptosis, and terminal differentiation of several tissues, including skeletal muscle, nervous system, and lens. Previous cell culture-based experiments have suggested that the retinoblastoma protein (pRb) and Ras operate in a common pathway to control cellular differentiation. Here we have tested the hypothesis that the proto-oncogene N-ras participates in Rb-dependent regulation of differentiation by generating and characterizing murine embryos deficient in both N-ras and Rb. We show that deletion of N-ras rescues a unique subset of the developmental defects associated with nullizygosity of Rb, resulting in a significant extension of life span. Rb؊/؊;N-ras؊/؊ skeletal muscle has normal fiber density, myotube length and thickness, in contrast to Rb-deficient embryos. Additionally, Rb؊/؊;N-ras؊/؊ muscle shows a restoration in the expression of the late muscle-specific gene ,MCK, and this correlates with a significant potentiation of MyoD transcriptional activity in Rb؊/؊;N-ras؊/؊ ؊/؊ ؊/؊ ؊/؊

compared to Rb myoblasts in culture. The improved differentiation of skeletal muscle in Rb ;N-ras on October 13, 2019 by guest embryos occurs despite evidence of deregulated proliferation and apoptosis, as seen in Rb-deﬁcient animals. Our ﬁndings suggest that the control of differentiation and proliferation by Rb are genetically separable.

The loss of tumor suppressor gene function is a common through its interaction with E2F, and its association with other event in the development of human cancer. The retinoblas- factors involved in altering chromatin structure (e.g., histone toma gene (Rb) has served as the paradigm for the study of this deacetylase and BRG1) leads to active repression of genes class of genes (67, 68). Mutations resulting in the inactivation involved in cell proliferation (e.g., cyclin E). The ability of pRb to of Rb are found in a large fraction of human cancers of both influence cell death also appears to involve its regulation of mesenchymal and epithelial origin (57, 62). An understanding E2F (13, 56, 63). of how the retinoblastoma protein (pRb) exerts its tumor- How pRb regulates differentiation is poorly understood. suppressive action can be gained from knowledge of the bio- Most differentiation programs involve withdrawal from the cell logical and molecular consequences of its inactivation. cycle, but the participation of pRb appears to extend beyond pRb participates in the control of cell cycle progression, an ability to merely facilitate the process by inhibiting E2F and apoptosis, and differentiation. How it exerts effects on prolif- cell cycle progression. Naturally occurring mutants of pRb can eration is well understood. This appears to be achieved by its be identified that retain tumor suppressor activity and the regulated interaction with the E2F family of transcription fac- ability to promote differentiation when assayed in vitro despite tors (14, 56, 63). E2F can bind to and promote the expression having lost the capability to bind to E2F (28, 58). Consistent of a number of genes involved in cell cycle progression (e.g., with this, pRb influences the activity of a number of transcrip- DHFR). pRb, by binding to E2F, can inhibit the transactivation tion factors known to participate in different differentiation function of E2F. In addition, pRb is targeted to promoters processes, such as MyoD, the glucocorticoid receptor, CBFA1, and C/EBP␤ (3, 4, 11, 43, 44, 59, 61). Also, cell culture studies * Corresponding author. Mailing address: Department of Medical have demonstrated a key role for pRb in myogenesis, osteo- Oncology and Medicine, Dana-Farber Cancer Institute and Harvard genic differentiation, and adipogenesis (3, 6, 11, 43, 54, 58, 61). Medical School, Boston, MA 02115. Phone: (617) 632-2206. Fax: (617) Mouse genetics have been employed to better understand 632-5417. E-mail: [email protected]. the physiological functions of pRb (35, 66). Mice heterozygous † Present addresses: Department of Molecular Oncology, Kyoto Uni- for Rb succumb to pituitary tumors (15, 17, 19), while inacti- versity Graduate School of Medicine, Sakyo-ku, Kyoto 606-8501, Japan. ‡ Present address: Chung-buk National University, College of Med- vation of both Rb alleles results in embryos that die in mid- icine, Cheong-Ju, Korea. gestation (5, 19, 31, 71). These embryos are characterized by

5256 VOL. 23, 2003 GENETIC INTERACTION BETWEEN Rb AND N-ras 5257 defects in erythroid, neuronal, and skeletal muscle differenti- and cell death. Our analyses suggest that N-ras operates with ation, and ectopic S-phase entry and apoptosis are observed in Rb in the control of cellular differentiation. the central nervous system (CNS), peripheral nervous system (PNS), lens, and skeletal muscle (5, 19, 31, 32, 71). The con- MATERIALS AND METHODS tribution of deregulated E2F activity to these phenotypes has Mouse strains. Parental Rbϩ/Ϫ and N-rasϩ/Ϫ mice were maintained on a been assessed with compound embryos lacking Rb and E2f-1 or mixed genetic background (C57BL/6 ϫ 129/Sv and C57BL/6 ϫ 129/Ola, respec- ϩ/Ϫ ϩ/Ϫ E2f-3 (21, 64, 72). These embryos live longer than their RbϪ/Ϫ tively) and intercrossed to generate subsequent founders. Rb ;N-ras females were crossed with Rbϩ/Ϫ;N-rasϩ/ϩ, Rbϩ/Ϫ;N-rasϩ/Ϫ,orRbϩ/Ϫ;N-rasϪ/Ϫ counterparts, and this has been attributed to a partial restora- males. Timed pregnancies were established by the detection of a plug, taken as tion of fetal liver erythropoiesis. Additionally, loss of either embryonic day 0.5 (E0.5). Mice and embryos were genotyped by PCR with E2f-1 or E2f-3 can suppress the deregulated proliferation and genomic DNA extracted from tails and yolk sacs, respectively, as previously apoptosis to significant but varying degrees in the CNS, PNS, described (19, 65). All animal experimentation was performed at the Dana- Farber Cancer Institute Animal Resource Facility in accordance with the guide- and lens. Importantly, the lens of Rb-deficient embryos also lines of the National Institutes of Health. shows signs of aberrant differentiation that are not rescued by Histology and immunohistochemistry. Embryos were fixed in Bouin’s solution, Downloaded from loss of E2f-1, suggesting again that Rb’s influence on cell cycle rinsed with 70% ethanol, and embedded in paraffin for sectioning. Sections (6 progression and apoptosis is genetically separable from its ␮m) were stained with hematoxylin and eosin (H&E). Alternatively, sections regulation of differentiation (36). A notable aspect of these were incubated with a monoclonal antibody (MY-32; Sigma) to myosin heavy chain (MHC) following deparaffinization and rehydration. To identify prolifer- Ϫ/Ϫ studies is the observation that the extended life span of Rb ; ating cells, bromodeoxyuridine (BrdU) was injected intraperitoneally (33 ␮g per Ϫ Ϫ Ϫ Ϫ Ϫ Ϫ E2f-1 / and Rb / ; E2f-3 / embryos reveals additional phe- mouse) 1 h prior to sacrifice. Fixed embryos were rinsed with 70% ethanol and notypes, including developmental defects in the lung and embedded in paraffin, from which sections were cut. After rehydration, the heart, and more pronounced defects in skeletal muscle. Ab- endogenous peroxidase activity was quenched with 3% H2O2–10% methanol in phosphate-buffered saline (PBS) (pH 7.4). Sections were then treated succes- normalities in skeletal muscle differentiation are also observed sively with 0.05 mM trypsin, 2 N HCl, and PBS (pH 6.0). After blocking with 6% in Rb-deficient mice with a partial reconstitution of Rb (71) goat serum, sections were incubated with an anti-BrdU mouse monoclonal an- http://mcb.asm.org/ and in RbϪ/Ϫ; Id2Ϫ/Ϫ mice (30). Indeed, respiratory failure tibody (B44; Becton Dickinson) in the presence of 0.5% Tween 20 in PBS (pH resulting from the complete lack of muscle fibers in the dia- 7.4). Bound primary antibody (MHC and BrdU) was detected with the ABC mouse peroxidase detection system (Santa Cruz Biotechnology). For the analysis phragm is thought to be responsible for their neonatal death. of cell death, embryos were fixed in formalin (3.7% formaldehyde in PBS), from Thus, Rb appears to play important roles in differentiation of which tissue sections were generated. Apoptosis was measured by terminal de- several tissues throughout embryonic development. Genetic oxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) dissection of Rb-dependent pathways holds promise for a assay with the ApopTag mouse peroxidase plus system (Intergen). Counterstain- ing was with 0.5% methyl green in 0.1 M sodium acetate (pH 4.0) or methanol. greater understanding of its role in development and may shed Analysis of gene expression. RNA was extracted from homogenized carcasses light on how its inactivation contributes to the genesis of tu- of live E14.5 embryos by using the RNeasy minikit (Qiagen). Northern blot

mors of diverse histological origin. analyses for MyoD and myogenin were performed as described previously (60). on October 13, 2019 by guest The ras genes are the most frequently mutated proto-onco- Radiolabeled antisense riboprobes were prepared by using the Lig’nScribe and MAXIscript in vitro transcription kits (Ambion). Multiplex RNase protection genes in human cancer (1, 24). Like pRb, Ras is thought to play assays were performed with RPAIII (Ambion) in accordance with the manufac- a critical role in differentiation and proliferation, and there has turer’s instructions. Probes correspond to the following sequences: muscle crea- been considerable progress in elucidating the downstream ef- tine kinase (MCK), nucleotides 808 to 1283 of the sequence under GenBank ␤ fector pathways through which Ras exerts its biological effects accession number X03233; and -actin, nucleotides 739 to 989 of the sequence under GenBank accession number X03672. (39, 40). Further, many of the transcription factors influenced MEFs and transcriptional transactivation assays. Mouse embryo fibroblasts by pRb, e.g., MyoD, glucocorticoid receptor, and C/EBP␤, are (MEFs) were prepared from live E12.5 embryos as described previously (9) and also influenced by Ras (16, 26, 27, 42, 45, 48–50, 55). Recent plated in six-well dishes at 105 cells per well in Dulbecco’s modified Eagle’s efforts have been devoted to understanding the individual roles medium (DMEM) containing 20% fetal bovine serum. MEFs were transfected as described in the legend to Fig. 3, using Fugene 6 (Roche). At 24 h after trans- of the three ras genes (H-ras,K-ras, and N-ras) in the mouse. fection, the cell medium was changed to DMEM containing 2% horse serum Mice nullizygous for H-ras or N-ras, or both, are developmen- (Sigma) and 10 ␮g of insulin per ml. Luciferase and ␤-galactosidase activities ϩ Ϫ tally normal (8, 18, 65). K-ras / mice are also developmen- were assayed 72 h later (33). The plasmids pCSA-MyoD (43), MCK-Luc (44), tally normal, but homozygous deletion of K-ras results in MEF2-luc (44), pCXN2-H-rasV12 (52), pBPJTR2-Rb (33), and pCMV-␤-gal (29) have been described previously. pBabe-N-ras was constructed by PCR lethality during gestation (22, 25). A role for N-ras during amplification with primers 5Ј-CGCGGATCCGCCACCATGACTGAGTACAA ϩ/Ϫ embryogenesis was revealed by the observation that K-ras ; ACTGGTGG-3Ј and 5Ј-CCGGAATTCCGGTTACATCACCACACATGGCA Ϫ Ϫ N-ras / embryos die during gestation, suggesting that K- and ATCCC-3Ј and pZIPNeoSV(X)I-EE-N-ras (23) as a template, followed by di- N-ras have partially overlapping functions (22). Testing for gestion with BamHI and EcoRI. The sequence was confirmed after subcloning into pBabe-puro. genetic interactions suggests a means of revealing other func- Infections and immunostaining. MEFs were infected with medium containing tional roles for N- and H-ras. MyoD virus from pBabe-MyoD (43)-transfected EcoPack-293 cells (Clon- We demonstrated previously that cultured fibroblasts de- tech/BD Biosciences). The viral titer was estimated from the efficiency of colony rived from Rb-deficient embryos possess abnormally high lev- formation following selection with 4 ␮g of puromycin per ml. Two thousand CFU 5 els of Ras activity and showed that the ability of pRb to neg- of virus was used to infect 10 cells per six-well dish in the presence of 20% fetal bovine serum. At 24 h after infection, the medium was changed to DMEM atively regulate Ras was linked to its effects on differentiation containing 2% horse serum plus 10 ␮g of insulin per ml. At 72 h postinfection, (33). To test the physiological relevance of these observations, cells were fixed in 4% paraformaldehyde in PBS, permeabilized with 0.1% Triton we have generated embryos lacking both Rb and N-ras.Wefind X-100 in PBS, and blocked with 3% bovine serum albumin (BSA) in PBS. MyoD that loss of N-ras reverses many of the differentiation defects was detected with rabbit serum to MyoD (M-318; Santa Cruz Biotechnology) and fluorescein isothiocyanate-conjugated goat anti-rabbit immunoglobulin G (Jack- observed in both mid- and late-gestational Rb-deficient em- son ImmunoResearch Laboratories) diluted 1:200 and 1:1,000, respectively, in bryos, despite the absence of obvious effects on proliferation 3% BSA in PBS. MHC was detected by using a monoclonal antibody to MHC 5258 TAKAHASHI ET AL. MOL.CELL.BIOL.

TABLE 1. Viability of embryos

No. of live embryos recovered (no. of nonviable embryos)a for the following mouse genotypeb Embryonic ϩ ϩ ϩ ϩ ϩ ϩ Ϫ Ϫ Ϫ Ϫ ϩ ϩ Ϫ Ϫ ϩ Ϫ Ϫ Ϫ Ϫ Ϫ stage Rb / ;N-ras / Rb / ;N-ras / Rb / ;N-ras / Rb / ;N-ras / Rb / ;N-ras / (a, b) (b, c) (a, b) (b, c) (b, c) E12.5 9 5 7 (3) [5 (2) (a), 2 (1) (b)] 8 (b) (c) 6 (b) (c) E13.5 11 8 7 (6) [4 (4), 3 (2) (b)] 9 (3) [6 (2) (b), 3 (1) (c)] 7 (b) (c) E14.5 14 7 6 (11) [2 (4) (a), 4 (7) (b)] 7 (10) [6 (8) (b), 1 (2) (c)] 9 (1) [7 (2) (1) (b), (c)] E15.5 7 12 (8) (a) 4 (12) [3 (9) (b), 1 (3) (c)] 12 (3) 7 (2) (b), 5 (1) (c) E16.5 6 8 (6) 6 (5) 8 (3) [4 (2) (b), 4 (1) (c)] E17.5 2 4 (3) (5) 2 (4) [1 (2) (b), 1 (2) (c)] E18.5 3 3 NDc (4) (3)

a Numbers in brackets show breakdown as a function of the particular cross used. b Genotypes of mice used for crosses are as follows: a, Rbϩ/Ϫ;N-rasϩ/Ϫ ϫ Rbϩ/Ϫ;N-rasϩ/ϩ;b,Rbϩ/Ϫ;N-rasϩ/Ϫ ϫ Rbϩ/Ϫ;N-rasϩ/Ϫ;c,Rbϩ/Ϫ;N-rasϩ/Ϫ ϫ Rbϩ/Ϫ; Ϫ Ϫ N-ras / . Downloaded from c ND, not determined.

Ϫ Ϫ (MY-32; Sigma) and rhodamine-conjugated goat anti-mouse immunoglobulin G Rb / embryos showed a dramatic reduction in fiber density (Jackson ImmunoResearch Laboratories) diluted 1:200 and 1:1,000, respectively, compared to that from wild-type and N-rasϪ/Ϫ embryos upon in 3% BSA in PBS. DAPI (4Ј,6Ј-diamidino-2-phenylindole) was included in the antifading mounting solution (Vectashield; Vector). H&E staining of tissue sections (Fig. 2A to C). This defect was even more apparent in sections immunostained with an antibody to MHC (Fig. 2E to G and M). Myotubes were also found RESULTS to be shorter and thinner in Rb-deficient skeletal muscle (Fig. http://mcb.asm.org/ Ϫ/Ϫ Ϫ/Ϫ Survival of embryos. We first determined if deletion of N-ras 2G and N). Strikingly, Rb ;N-ras axial muscle clearly prolonged the survival of Rb-deficient embryos. Rb and N-ras displayed normal fiber density, myotube length, and thickness heterozygotes were bred, and the resulting Rbϩ/Ϫ;N-rasϩ/Ϫ (Fig. 2D, H, M, and N). The presence of abnormal large nuclei Ϫ/Ϫ Ϫ/Ϫ associated with ectopic DNA synthesis was detected at low mice were intercrossed. No viable Rb ;N-ras pups Ϫ Ϫ ϩ Ϫ ϩ Ϫ ϳ / were recovered. Subsequently, Rb / ;N-ras / females were frequency ( 1 to 2%) by H&E and BrdU staining in Rb ; Ϫ/Ϫ Ϫ/Ϫ crossed with Rbϩ/Ϫ;N-rasϩ/ϩ, Rbϩ/Ϫ;N-rasϩ/Ϫ,orRbϩ/Ϫ; N-ras and Rb muscle, consistent with previous findings N-rasϪ/Ϫ males, and embryonic viability during a time course (Fig. 2D and O and data not shown) (21, 71). Additionally, apoptotic cells (i.e., TUNEL positive) were seen in the skeletal of gestation was assessed by the detection of a beating heart Ϫ Ϫ Ϫ Ϫ

/ / on October 13, 2019 by guest (Table 1). Death of embryos lacking Rb alone was first noted at muscle of Rb-deficient and Rb ;N-ras embryos, unlike Ϫ/Ϫ E12.5 (30%). Consistent with previous results, the majority of their wild-type and N-ras counterparts (Fig. 2I to L and P). Rb-deficient embryos (65%) were dead by E14.5, with no via- Together, these results suggest that loss of N-ras prevents the ble embryos being recovered at E15.5 or beyond. Remarkably, reduction in muscle fiber density and abnormal myotube for- most RbϪ/Ϫ;N-rasϪ/Ϫ embryos (73%) were alive at E16.5, with mation seen in E14.5 Rb-deficient skeletal muscle, while having some living as long as E17.5 (33%). RbϪ/Ϫ;N-rasϩ/Ϫ embryos no impact on ectopic proliferation or cell death. Ϫ/Ϫ Ϫ/Ϫ showed an intermediate level of survival. RbϪ/Ϫ;N-rasϪ/Ϫ em- Given the relatively normal appearance of Rb ;N-ras bryos were macroscopically indistinguishable from their wild- skeletal muscle at E14.5, we extended our gross histological type littermates at E13.5 (Fig. 1A and B). RbϪ/Ϫ;N-rasϪ/Ϫ examination to the expression of muscle-specific genes. For embryos continued to display a remarkably normal appearance this analysis, total RNA derived from the carcasses of live at a later stage in development (E15.5), although they were E14.5 embryos was used. MyoD and myogenin, both of which noticeably smaller (Fig. 1D and E). N-rasϪ/Ϫ embryos were are expressed during early stages of muscle development (53), ϩ Ϫ ϩ Ϫ ϩ Ϫ indistinguishable in appearance from their wild-type litter- were expressed at comparable levels in Rb / ;N-ras / , Rb / ; Ϫ Ϫ Ϫ Ϫ ϩ Ϫ Ϫ Ϫ Ϫ Ϫ mates (data not shown). Together, these data indicate that loss N-ras / , Rb / ;N-ras / , and Rb / ;N-ras / embryos as of N-ras can significantly prolong the life span of Rb-deficient determined by Northern blot analysis (Fig. 2Q). MCK, a late embryos, suggesting the possibility that a genetic interaction marker of muscle differentiation, is normally expressed from E13 between these genes may reverse key developmental defects onward (38). RNase protection assay for MCK revealed almost no Ϫ Ϫ ϩ Ϫ associated with Rb loss. expression in Rb-deficient (Rb / ;N-ras / ) embryos (Fig. 2R), Skeletal muscle development. Our previous cell culture- consistent with a previous report on Rb-deficient embryos with based work linked pRb and Ras to the control of myogenic partial reconstitution of Rb (71). By contrast, normal levels of Ϫ Ϫ Ϫ Ϫ differentiation, and thus we initially focused on the analysis of MCK transcripts were detected in Rb / ;N-ras / embryos. skeletal muscle. Rb-deficient embryos display a number of de- MCK is a transcriptional target of MyoD, and the activity of fects in skeletal muscle differentiation, many of which are ob- this transcription factor requires pRb for its full activation served as early as E13.5, and these are exacerbated in late (44). In this context, pRb acts in part by cooperating with gestational Rb compound mutants (see the introduction). We MyoD to promote the transcriptional activity of MEF2C, began by assessing whether N-ras loss might influence the which also participates in the transcriptional induction of MCK development of Rb-deficient muscle by directly comparing the (44). We therefore assessed the influence of N-ras loss on the axial muscle from live E14.5 wild-type, N-rasϪ/Ϫ, RbϪ/Ϫ, and activity of MyoD and MEF2C in cells nullizygous for Rb. RbϪ/Ϫ;N-rasϪ/Ϫ littermates. Thoracic skeletal muscle from MEFs were generated from wild-type, Rbϩ/Ϫ,N-rasϪ/Ϫ, RbϪ/Ϫ, VOL. 23, 2003 GENETIC INTERACTION BETWEEN Rb AND N-ras 5259 Downloaded from http://mcb.asm.org/

FIG. 1. Effects of N-ras loss on the appearance of Rb-deficient embryos. Embryos from the same litter with the indicated genotype were recovered at E13.5 (A to C) and E15.5 (D to F) and immediately photographed in saline. Bars, 150 ␮m. on October 13, 2019 by guest and RbϪ/Ϫ;N-rasϪ/Ϫ embryos and transfected with a MyoD- these results suggest that restoration of MCK expression fol- encoding plasmid, and the activity of an MCK promoter-re- lowing loss of N-ras in Rb-deficient skeletal muscle in the porter construct was measured under culture conditions known mouse correlates with a cell-autonomous reversal in the defect to induce myogenic differentiation. The activity of MyoD was in MyoD and MEF2C function in vitro. significantly reduced in Rb-deficient myoblasts. By contrast, The analyses described above provided a direct compari- RbϪ/Ϫ;N-rasϪ/Ϫ myoblasts possessed levels of activity similar son between wild-type, RbϪ/Ϫ, and RbϪ/Ϫ;N-rasϪ/Ϫ skeletal to those seen in wild-type, Rbϩ/Ϫ, and N-rasϪ/Ϫ cells (Fig. 3A). muscle from matched littermates. The next question was Similar observations were found with a MEF2 promoter-re- whether the improved skeletal muscle development ob- porter construct (Fig. 3B). To rule out the possibility that served in E14.5 RbϪ/Ϫ;N-rasϪ/Ϫ embryos persisted during N-ras-independent mechanisms might be responsible for the later stages of embryonic development. We found that the restoration of MyoD and MEF2C activity in RbϪ/Ϫ;N-rasϪ/Ϫ lengths and thicknesses of myotubes in RbϪ/Ϫ;N-rasϪ/Ϫ and MEFs, we reconstituted N-ras expression. Expression of wild- wild-type thoracic muscle from sagittal sections of E17.5 type N-ras in RbϪ/Ϫ;N-rasϪ/Ϫ, but not Rbϩ/Ϫ, MEFs signifi- embryos were largely indistinguishable as judged by MHC cantly reduced the activity of both the MCK (Fig. 3C) and immunostaining (Fig. 4A and B). A similar analysis of trans- MEF2 (Fig. 3D) reporters, suggesting that loss of N-ras and verse sections though the cervical skeletal muscle revealed not other genetic events potentiates the activity of MyoD and that the densities of the fibers in RbϪ/Ϫ;N-rasϪ/Ϫ and wild- MEF2C in Rb-deficient myoblasts. Constitutively active onco- type muscle were also similar (Fig. 4C and D). However, the genic H-Ras inhibited transcriptional activity in Rbϩ/Ϫ myo- fibers appeared to be somewhat disorganized in RbϪ/Ϫ; blasts (Fig. 3C and D), consistent with previous reports (48, N-rasϪ/Ϫ muscle. Nevertheless, from the area occupied by 49). Lastly, we explored the notion that the influence of N-ras the fibers in transverse sections and the length of the myo- loss on myogenesis was cell autonomous. To this end, wild- tubes seen in sagittal sections, these data suggest that the type, RbϪ/Ϫ, and RbϪ/Ϫ;N-rasϪ/Ϫ MEFs were incubated with axial muscle masses (proportional to body size) were similar a MyoD-encoding retrovirus such that approximately 1% of in RbϪ/Ϫ;N-rasϪ/Ϫ and wild-type embryos. Normal fi- the cells were infected. Coimmunostaining for MyoD and ber density and MHC staining were also seen in intercostal MHC revealed that the majority of cells that express MyoD muscle from sagittal sections of RbϪ/Ϫ;N-rasϪ/Ϫ embryos also express MHC and form myotubes in wild-type and RbϪ/Ϫ; (Fig. 4E and F). A similar analysis of the diaphragm re- N-rasϪ/Ϫ, but not RbϪ/Ϫ, myoblasts (Fig. 3E to K). Together, vealed normal levels of MHC, although a slightly reduced 5260 TAKAHASHI ET AL. MOL.CELL.BIOL. Downloaded from http://mcb.asm.org/ on October 13, 2019 by guest FIG. 2. Effect of N-ras loss on skeletal muscle development in E14.5 Rb-deficient embryos. (A to H) Longitudinal sections through the fibers of thoracic somite-associated skeletal muscle from sagittal sections of E14.5 embryos derived from the same litter with the indicated genotype were stained with H&E (A to D) or immunostained by using an antibody to MHC (E to H). Large nuclei (arrow) in myotubes are indicated in panel D. Magnification, ϫ40. (I to L) Apoptosis (TUNEL) observed in myoblasts of thoracic skeletal muscle of E14.5 embryos of the indicated genotype. Magnification, ϫ60. (M) The density of MHC in myotubes stained with antibody to MHC was quantified by using the NIH image 1.61 program. The strength of the signal was read as pixel number per square micrometer. Ten myotubes per embryo were analyzed, and the average density of the MHC signal Ϯ standard error was calculated. In parentheses is the number of embryos used for the analysis. (N) The length of myotubes immunostained with an antibody to MHC was quantified. Longitudinal sections of thoracic skeletal muscle from E14.5 embryos were analyzed by microscopic observation. Twenty myotubes per embryo were measured, and average length Ϯ standard error is presented. In parentheses is the number of embryos used for the analysis. (O) One hundred myotubes in the thoracic skeletal muscle were analyzed for the presence of giant nuclei following staining with H&E. The average percentage Ϯ standard error is presented. In parentheses is the number of embryos used for the analysis. (P) The level of apoptosis was quantified by counting the frequency of TUNEL-positive cells per 300 nuclei analyzed in the thoracic muscle. The average percentage Ϯ standard error is presented. In parentheses is the number of embryos used for the analysis. (Q) The levels of MyoD and myogenin transcripts in total RNA derived from carcasses of live E14.5 embryo littermates of the indicated genotype were determined by Northern blot analysis. (R) Expression of MCK determined by RNase protection assay with RNAs derived from carcasses of live E14.5 embryos of the indicated genotype from two sets of matched littermates, with lanes 1, 3, 5, and 7 showing data from one litter and lanes 2, 4, and 6 showing data from the other litter. The ratio of MCK to ␤-actin expression is shown at the bottom of the panel.

fiber density was apparent (Fig. 4G to J). The presence of dant in the intermediate zone of the hindbrain (CNS) in both cells with abnormally large nuclei still persisted in RbϪ/Ϫ; RbϪ/Ϫ and RbϪ/Ϫ;N-rasϪ/Ϫ, but not wild-type or N-rasϪ/Ϫ, N-rasϪ/Ϫ skeletal muscle (Fig. 4K and L). These results embryos (Fig. 5A to D and I). Similar results were obtained suggest that loss of N-ras allows for near-normal develop- from our analysis of the fiber cell compartment of the development of Rb-deficient skeletal muscle during embryogenesis. ing lens and dorsal root ganglion of the PNS (Fig. 5I and data Ectopic S-phase entry and death in the CNS. E13.5 RbϪ/Ϫ not shown). Apoptosis was assessed by TUNEL staining. As embryos are characterized by extensive proliferation and cell described previously, RbϪ/Ϫ embryos showed an elevated level death in normally postmitotic regions of the CNS, PNS, and of cell death in the lens, the PNS, and the cortical region lens (5, 19, 31). We therefore sought to determine if loss of around the fourth ventricle of the brain (CNS) compared to N-ras affected these phenotypes. Pregnant females at 13.5 days wild-type and N-rasϪ/Ϫ embryos (Fig. 5E to G and J and data of term were injected intraperitoneally with BrdU, and DNA not shown). RbϪ/Ϫ;N-rasϪ/Ϫ mutants showed a degree of apo- synthesis was assessed by immunological detection of incorpo- ptosis similar to that found in Rb-deficient embryos (Fig. 5G, rated BrdU in tissue sections. BrdU-positive cells were abun- H, and J and data not shown). Together, these results indicate VOL. 23, 2003 GENETIC INTERACTION BETWEEN Rb AND N-ras 5261 Downloaded from http://mcb.asm.org/ on October 13, 2019 by guest

FIG. 2—Continued. that the ectopic proliferation and apoptosis characteristic of oles and primitive alveoli were not observed in embryos lacking Rb-deficient embryos are not altered by the concomitant loss of Rb and either E2f-1 or E2f-3. Our analysis of E17.5 RbϪ/Ϫ; N-ras. N-rasϪ/Ϫ embryos revealed a normal appearance for the lung Lung and heart development. Given the improved survival (Fig. 6A and B). Consistent with these findings, the space oc- and development of RbϪ/Ϫ;N-rasϪ/Ϫ embryos, we conducted a cupied by the lung in the intrathoracic cavity of RbϪ/Ϫ;N-rasϪ/Ϫ full histological analysis of these animals. We focused initially embryos was comparable to that observed in wild-type littermates on the lung, since two previous reports had revealed an essen- (data not shown). In addition, the hearts of RbϪ/Ϫ; E2f-3Ϫ/Ϫ tial role for Rb in the development of this organ during late embryos have been reported to possess reduced cardiac muscle gestation (64, 72). Specifically, well-defined terminal bronchi- fiber density, resulting in a marked thinning of the heart wall 5262 TAKAHASHI ET AL. MOL.CELL.BIOL. Downloaded from http://mcb.asm.org/ on October 13, 2019 by guest FIG. 3. Effect of N-ras loss on MyoD transcriptional activity in Rb-deficient myoblasts. (A) MEFs of the indicated genotypes were transfected with an MCK promoter-reporter construct (MCK-luc; 0.25 ␮g), pCSA-MyoD (1.25 ␮g), and pCMV-␤-gal (0.25 ␮g). Twenty-four hours later the cells were allowed to differentiate for 48 h. Luciferase and ␤-galactosidase were determined, and normalized fold activations were calculated relative to the corrected luciferase activity in the absence of MyoD. Results are the means Ϯ standard errors for three independent experiments performed in triplicate. The numbers at the top are for the particular MEFs used. Groups 1 and 5; 2 and 6; and 3, 4, 7, and 8 are each derived from different litters. (B) MEFs (1, 4, 5, and 7; designations are as described for panel A) were transfected as described for panel A except that a MEF2-luc reporter was used. Fold activations were calculated relative to the corrected luciferase activity in the absence of MyoD. Results are the means Ϯ standard errors for three independent experiments performed in triplicate. (C) MEFs (3 and 7; designations are as described for panel A) were transfected as described for panel A. As indicated, included in the transfections were plasmids encoding pRb (pBPJTR2-Rb; 0.25 ␮g), N-ras (pBabe-N-ras; 0.5 ␮g), or H-rasV12 (pCXN2-H-rasV12; 0.5 ␮g). Fold activations were calculated relative to the corrected luciferase activity in the absence of MyoD. Results are the means Ϯ standard errors for three independent experiments performed in triplicate. (D) MEFs (3 and 7; designations are as described for panel A) were transfected as described for panel B. As indicated, included in the transfections were plasmids encoding N-ras (pBabe-N-ras; 0.5 ␮g) or H-rasV12 (pCXN2-H-rasV12; 0.5 ␮g). Fold activations were calculated relative to the corrected luciferase activity in the absence of MyoD. Results are the means Ϯ standard errors for three independent experiments performed in triplicate. (E to J) MEFs (3, 5, and 7; designations are described for panel A) were infected with a MyoD-encoding retrovirus. The quantity of retrovirus-containing medium was adjusted such that approximately 1% of the cells formed myotubes in wild-type MEFs. Following differentiation, immunofluorescence was performed for MyoD (with fluorescein isothiocyanate) and MHC (with rhodamine). Nuclei were stained with DAPI. (K) Quantification of the results shown in panels E to G. The percentage of MyoD-positive cells that also showed strong staining for MHC was determined by analyzing equivalent numbers of total cells (ϳ1,000) for each genotype in 10 fields (magnification, ϫ10). The average percentage Ϯ standard error is presented. All cells showing strong MHC staining were also MyoD positive.

(72). This defect was not apparent in RbϪ/Ϫ;N-rasϪ/Ϫ embryos attempts to rescue this embryonic lethality have revealed that (Fig. 6C and D). Analysis of other organs did not reveal any Rb is also required at later stages in development in several defects beyond those described for Rb-deficient embryos. tissues. The diversity of defects exhibited in Rb-deficient embryos suggests that pRb engages several downstream effector DISCUSSION pathways to affect a multitude of biological outcomes. Our results demonstrate that loss of N-ras significantly extends the To understand the functions of Rb, much effort has been life span of Rb-deficient embryos and that the effect of N-ras devoted to characterizing the biological consequences of its loss on the phenotypes associated with Rb nullizygosity extends inactivation in the mouse. RbϪ/Ϫ embryos die in midgestation to several differentiation programs, including skeletal muscle with defects in a restricted set of tissues. Results from various differentiation and possibly development of the lung and heart. VOL. 23, 2003 GENETIC INTERACTION BETWEEN Rb AND N-ras 5263 Downloaded from http://mcb.asm.org/ on October 13, 2019 by guest

FIG. 3—Continued.

Below we discuss the effects of N-ras loss on the phenotypes of RbϪ/Ϫ; Id2Ϫ/Ϫ neonates, suggesting that these mice also associated with deficiency in Rb. died of respiratory failure (30). The lack of an effect of Id2 loss Skeletal muscle differentiation. The first extensive analysis on any of the muscle phenotypes is not surprising given that it of Rb-deficient skeletal muscle was made by using RbϪ/Ϫ em- is not expressed in this tissue. This, however, is not the case for bryos in which Rb expression had been partially restored (21, E2f-1 and E2f-3. The inability of E2f-1 or E2f-3 deficiency to 71). Importantly, the transgene used, although effective in ex- ameliorate any of the muscle phenotypes, while clearly rescu- tending life span, does not direct expression of Rb to the ing defects in other tissues, has led to the notion that the muscle (20), affording those workers the ability to study skel- pathway(s) by which pRb influences skeletal muscle develop- etal muscle differentiation in the absence of Rb at later stages ment may involve a positive differentiation function of pRb of gestation. Defects in skeletal muscle were readily apparent (64). Consistent with this, in vitro studies indicate that the at the time that Rb-deficient embryos normally die, and they transcriptional induction of certain muscle-specific genes by became more pronounced at later stages of gestation, during MyoD requires Rb (11, 43). However, with the myriad of de- which time the skeletal muscle continues to develop. Rb-defi- fects observed in RbϪ/Ϫ skeletal muscle in vivo, it is unclear cient skeletal muscle was characterized by reduced fiber den- whether there is a single underlying mechanism. sity, abnormal myotube formation, and length and lack of Our results begin to address these issues. Loss of N-ras expression of late markers of differentiation. Additionally, apo- significantly improved myotube formation and muscle fiber ptotic cells and ectopic S-phase entry with evidence of en- density in both mid- and late-gestational Rb-deficient muscle. doreduplication were noted. So severe was the muscle defect In addition, the expression of a late marker of differentiation, that respiratory failure is thought to be the cause of death. MCK, was restored to wild-type levels. By contrast, cell death Similar skeletal muscle phenotypes were noted following loss and ectopic S-phase entry still persisted in RbϪ/Ϫ;N-rasϪ/Ϫ of Rb and E2f-1, E2f-3,orId2 (30, 64, 72). A complete lack of skeletal muscle. These results indicate that the various pheno- muscle fibers and MHC staining was noted in the diaphragms types that characterize Rb-deficient muscle are genetically sep- 5264 TAKAHASHI ET AL. MOL.CELL.BIOL. Downloaded from http://mcb.asm.org/ on October 13, 2019 by guest

FIG. 4. Effect of N-ras loss on skeletal muscle development in E17.5 Rb-deficient embryos. (A to D) Thoracic (A and B) and cervical (C and D) skeletal muscle, immunostained with an antibody to MHC and counterstained with methyl green, derived from sagittal sections of E17.5 embryos of the indicated genotype from the same litter. Panels A and B show longitudinal sections of the muscle, while panels C and D show transverse sections through the muscle fibers. Magnification, ϫ20. (E to J) Immunostained and counterstained sections of intercostal muscle between the fourth and fifth ribs (E and F) and the diaphragm (G to J) derived from sagittal sections of embryos of the indicated genotype. Represented are the transverse sections of each muscle group. Magnifications, ϫ20 (E and F), ϫ10 (G and H), and ϫ40 (I and J). (K and L) Longitudinal sections through the fibers of thoracic muscle from sagittal sections of E17.5 embryos derived from the same litter with the indicated genotype were stained with H&E. Note the presence of abnormal large nuclei in RbϪ/Ϫ;N-rasϪ/Ϫ muscle (arrow). Magnification, ϫ40. VOL. 23, 2003 GENETIC INTERACTION BETWEEN Rb AND N-ras 5265 Downloaded from http://mcb.asm.org/ on October 13, 2019 by guest

FIG. 5. Effects of N-ras loss on ectopic S-phase entry and apoptosis. (A to D) Transverse sections of the intermediate zone of the hindbrain from E13.5 embryos of the indicated genotype were stained for cells in S phase (with BrdU). Ventricular (v) and intermediate (i) zones are indicated. (E to H) Mid-sagittal sections of the cortical region around the fourth ventricle from E13.5 embryos of the indicated genotype were stained for apoptotic cells (with TUNEL). (I) The level of ectopic S phase was quantified by counting the frequency of BrdU-positive cells per unit area in tissue sections of the intermediate zone of the hindbrain (CNS, ectopic), dorsal root ganglia (PNS), and fiber compartment of lens. The total cell number was determined by counting cells counterstained with methyl green. The frequency for RbϪ/Ϫ samples was set to 1.0, and the relative ratio of BrdU-positive cells is shown. Values are means Ϯ standard errors for two to four embryos. (J) The level of apoptosis was quantified by counting the frequency of TUNEL-positive cells per unit area of tissue of the cortical region around the fourth ventricle of CNS, dorsal root ganglia of PNS, and fiber compartment of lens. The total cell number was determined by counting cells counterstained with methyl green. The frequency for RbϪ/Ϫ samples was set to 1.0, and the relative ratio of TUNEL-positive cells is shown. Values are means Ϯ standard errors for three to six embryos. arable. Further, they suggest that deregulated proliferation (48–50). These findings are consistent with our previous dem- and apoptosis are likely not responsible for the inability of onstration that aberrant activation of Ras following loss of Rb certain aspects of the muscle differentiation program to pro- is linked to the defect in MyoD transcriptional function in Rb- ceed during embryogenesis. Our data suggest that multiple deficient cells (33). Our genetic analysis in cell culture suggests Rb-dependent pathways influence skeletal muscle differentia- that loss of N-ras (effectively reducing the levels of active Ras) tion and that the genetic interaction between Rb and N-ras in Rb-deficient fibroblasts restores the activity of MyoD, which controls of a subset of these. participates in the induction of MCK during myogenesis. Fur- Several studies have documented that constitutively active ther, our results reveal that activity of MEF2C, which cooper- Ras can block the transcriptional functions of MyoD (26, 45), ates with MyoD in the transcription of the MCK gene (44), is and this appears to occur through multiple effector pathways also restored in RbϪ/Ϫ;N-rasϪ/Ϫ myoblasts. Together with our 5266 TAKAHASHI ET AL. MOL.CELL.BIOL. Downloaded from http://mcb.asm.org/

FIG. 6. Effect of N-ras loss on lung and heart development in E17.5 Rb-deficient embryos. (A and B) H&E-stained sections from lung tissue from E17.5 embryos of the indicated genotypes. Terminal bronchioles (b) and primitive alveoli (a) are indicated. Magnification, ϫ10. (C and D) H&E-stained left ventricular wall of heart from E17.5 embryos of the indicated genotypes. The bar indicates the thinnest part of the heart wall. Magnification, ϫ10.

observation that RbϪ/Ϫ;N-rasϪ/Ϫ embryos show normal levels Separation of differentiation and proliferation control. on October 13, 2019 by guest of MCK expression, these data suggest that the ability of N-ras Studies in mammalian cell culture have placed pRb both up- loss to rescue several of the skeletal muscle defects manifest in stream and downstream of Ras. Inhibition of Ras activity

Rb-deficient embryos is mediated at least in part through a brings about G1 arrest, and this is dependent on the presence cell-autonomous rescue of MyoD and MEF2C function. of functional pRb as well as E2F-4 and E2F-5 (10, 34, 41, 47). Recently, it has been shown that Rb-deficient placentas are In addition, ectopic expression of cyclin D1 and its catalytic abnormal and that this contributes to certain developmental partner Cdk4 can reverse the arrest induced by inhibition of defects that characterize RbϪ/Ϫ embryos (69). Specifically, Rb- Ras (47). Given that cyclin D1 is a transcriptional target of the deficient embryos supplied with wild-type extraembryonic cells Ras/mitogen-activated protein kinase (MAPK) pathway, it has can be carried to term and die soon after birth. Analyses of been suggested that pRb is a downstream target of Ras signal- these embryos revealed that the erythroid, CNS, and PNS ing. defects were largely rescued (69). By contrast, defects in cer- Inactivation of pRb by simian virus 40 large T antigen has tain tissues, such as skeletal muscle (69; G. Leone, personal been shown to result in elevated Ras activity (33, 51). In ad- communication) and the lens (69) were not rescued. Further, dition, Rb-deficient mouse embryonic fibroblasts possess ele- respiratory failure due to defects in the muscle fibers in the vated Ras activity compared to their wild-type counterparts diaphragm is responsible for the neonatal death of these ani- (33). Previously, we showed that the ability of pRb to regulate mals (G. Leone, personal communication). This suggests that Ras activity was linked to pRb’s positive control of certain Rb-dependent placental abnormalities do not contribute to the differentiation processes (33). For example, the activity of the skeletal muscle defects and thus the genetic interaction be- glucocorticoid receptor, which is known to require pRb for tween Rb and N-ras described here is confined to the embryo activation (59), was induced in Rb-deficient cells by the inhi- proper. Consistent with this interpretation, RbϪ/Ϫ embryos in bition of Ras activity. In this regard, it is interesting that Rb- which Rb expression has been partially restored by creation of deficient lungs lacking E2f-1 or E2f-3 show impaired develop- transgenic animals (71) or where there is a restoration of Rb ment, resulting in an atelectic appearance similar to that function in extraembryonic tissues (69) share many features, observed in glucocorticoid receptor-deficient mice (7, 64, 72). suggesting that in the former placental defects were not This phenotype is not observed in RbϪ/Ϫ;N-rasϪ/Ϫ embryos, present. However, in both of these genetically modified mice suggesting the possibility that loss of N-ras ameliorates the skeletal muscle defects are still manifest, indicating that ab- Rb-deficient lung defect through an influence on glucocorti- normalities in extraembryonic cell lineages do not contribute coid receptor transcriptional functions. Likewise, we reported to Rb-dependent skeletal muscle phenotypes. that inhibition of aberrant Ras activity in Rb-deficient myo- VOL. 23, 2003 GENETIC INTERACTION BETWEEN Rb AND N-ras 5267 blasts potentiated MyoD transcriptional activity and induced Rb. We suggest that the ability of Rb to function upstream of the expression of a late marker of differentiation (33). Impor- ras represents an evolutionarily conserved signaling pathway tantly, these in vitro findings are in agreement with our in vivo that impinges on several differentiation programs. analysis demonstrating that the expression of MCK, a tran- Ϫ/Ϫ Ϫ/Ϫ scriptional target of MyoD, is restored in Rb ;N-ras ACKNOWLEDGMENTS muscle and our observation that MyoD is more transcription- ally active in RbϪ/Ϫ;N-rasϪ/Ϫ MEFs than in their RbϪ/Ϫ coun- We express special thanks to C. McMahon, J. Lamb, M. Loda, D. Livingston, A. Lassar, G. Dranoff, S. Lux, P. Sicinski, W. Kaelin, J. terparts. More recently, others have linked the elevated Ras DeCaprio, K. Tsai, R. Costa, and A. Silva for help, advice, and en- and MAPK activity in Rb-deficient MEFs to their inability to couragement. We thank G. Leone for sharing results prior to publica- undergo adipose conversion (12). Together, these data suggest tion; M. McCagg for help with animal procedures; Z. Lee and M. Loda that pRb can act antagonistically upstream of Ras to influence for help with pathology; M. Ciemerych for help with BrdU assays; A. Lassar, W. Wright, and J. Jackson for plasmids; and J. Lamb, P. certain differentiation processes in vivo without an apparent Sicinski, W. Sellers, and M. Loda for critical review of the manuscript. effect on the cell cycle.

This work was supported by National Institutes of Health grant Downloaded from Studies with the nematode Caenorhabditis elegans have re- CA65842 to M.E.E. and by a Massachusetts Prostate Cancer Research vealed some striking similarities to the signaling between pRb Grant to C.T. C.T. was supported in part by the NCI-JFCR Scientist and Ras in mammalian cells. C. elegans Ras (LET-60) function Exchange Program. M.E.E. is a Leukemia and Lymphoma Society Scholar. is not essential for proliferation during embryogenesis and the four larval stages but is required later in development for the REFERENCES establishment of distinct cell fates (70). The C. elegans genes 1. Bos, J. L. 1989. ras oncogenes in human cancer: a review. Cancer Res. lin-35, eﬂ-1, and dpl-1 encode proteins similar to mammalian 49:4682–4689. pRb, E2F, and DP, respectively. Genetic epistasis tests indicate 2. Ceol, C. J., and H. R. Horvitz. 2001. dpl-1 DP and eﬂ-1 E2F act with lin-35 Rb to antagonize Ras signaling in C. elegans vulval development. Mol. Cell

that each of these proteins acts upstream of or in parallel to the 7:461–473. http://mcb.asm.org/ Ras/MAPK pathway during postembryonic vulval induction (2, 3. Chen, P.-L., D. J. Riley, Y. Chen, and W.-H. Lee. 1996. Retinoblastoma protein positively regulates terminal adipocyte differentiation through direct 37). No discernible effects on cell proliferation were observed. interaction with C/EBPs. Genes Dev. 10:2794–2804. In a related study, mutations in efl-1 and dpl-1 were shown to 4. Chen, P.-L., D. J. Riley, S. Chen-Kiang, and W.-H. Lee. 1996. Retinoblas- cause defects in embryonic polarity in a cell cycle-independent toma protein directly interacts with and activates the transcription factor NF-IL6. Proc. Natl. Acad. Sci. USA 93:465–469. manner (46). In addition, it was demonstrated that EFL-1 and 5. Clarke, A. R., E. R. Maandag, M. van Roon, N. M. T. van der Lugt, M. van DPL-1 exert their effects by negatively regulating MAPK ac- der Valk, M. L. Hooper, A. Berns, and H. te Riele. 1992. Requirement for a tivity, suggesting that EFL-1–DPL-1 complexes operate up- functional Rb-1 gene in murine development. Nature 359:328–330. 6. Classon, M., B. K. Kennedy, R. Mulloy, and E. Harlow. 2000. Opposing roles stream of the Ras/MAPK pathway (46). These data parallel of pRB and p107 in adipocyte differentiation. Proc. Natl. Acad. Sci. USA those for mammalian systems in that they identify an antago- 97:10826–10831. 7. Cole, T. J., J. A. Blendy, P. Monaghan, K. Krieglstein, S. W., A. Aguzzi, G. on October 13, 2019 by guest nism between pRb/E2F- and Ras-mediated signaling. They Fantuzzi, E. Hummler, K. Unsicker, and G. Schutz. 1995. Targeted disrup- also suggest that pRb functions upstream of Ras to influence tion of the glucocorticoid receptor gene blocks adrenergic chromaffin cell differentiation. development and severely retards lung maturation. Genes Dev. 9:1608–1621. 8. Esteban, L. M., C. Vicario-Abejon, P. Fernandez-Salguero, A. Fernandez- A question that arises when comparing the nematode and Medarde, N. Swaminathan, K. Yienger, E. Lopez, M. Malumbres, R. McKay, mammalian studies is whether E2F and Ras operate in com- J. M. Ward, A. Pellicer, and E. Santos. 2001. Targeted genomic disruption of mon or different pathways. Our results pertaining to the CNS, H-ras and N-ras, individually or in combination, reveals the dispensability of both loci for mouse growth and development. Mol. Cell. Biol. 21:1444–1452. lens, and skeletal muscle shed some light on this. Loss of E2f-1 9. Freshney, R. I. 2000. Culture of animal cells: a manual of basic technique, or E2f-3 significantly reverses the ectopic S-phase entry and 4th ed. Wiley-Liss, New York, N.Y. 10. Gaubatz, S., G. J. Lindeman, S. Ishida, L. Jakoi, J. R. Nevins, D. M. apoptosis observed in the CNS and lens of Rb-deficient em- Livingston, and R. E. Rempel. 2000. E2F4 and E2F5 play an essential role in bryos (64, 72), while loss of N-ras has no impact on these pocket protein-mediated G1 control. Mol. Cell 6:729–735. phenotypes. On the other hand, loss of N-ras can ameliorate 11. Gu, W., J. W. Schneider, G. Condorelli, S. Kaushal, V. Mahdavi, and B. Nadal-Ginard. 1993. Interaction of myogenic factors and the retinoblastoma many but not all of the abnormalities in Rb-deficient skeletal protein mediates muscle cell commitment and differentiation. Cell 72:309–324. muscle, while deficiency in E2f-1 or E2f-3 has no apparent 12. Hansen, J. B., R. K. Petersen, C. Jorgensen, and K. Kristiansen. 2002. effect (64, 72). Thus, in certain tissues a genetic interaction Deregulated MAPK activity prevents adipocyte differentiation in fibroblasts lacking the retinoblastoma protein. J. Biol. Chem. 277:26335–26339. between Rb and E2f-1 or E2f-3 is not apparent while that 13. Harbour, J. W., and D. C. Dean. 2000. Rb function in cell-cycle regulation between Rb and N-ras is and vice versa. These observations and apoptosis. Nat. Cell Biol. 2:E65–E67. 14. Harbour, J. W., and D. C. Dean. 2000. The Rb/E2F pathway: expanding roles suggest that N-ras and E2f do not operate in a common sig- and emerging paradigms. Genes Dev. 14:2393–2409. naling pathway but rather that the genetic interactions between 15. Harrison, D. J., M. L. Hooper, J. F. Armstrong, and A. R. Clarke. 1995. Rb and E2f-1 or -3 and between Rb and N-ras indicate a Effects of heterozygosity for the Rb-1t19neo allele in the mouse. Oncogene 20:1615–1620. bifurcation in Rb function. Consistent with this interpretation, 16. Hock, J. R., A. Ziemiecki, R. Klemenz, R. Friis, and B. Groner. 1989. cell culture-based experiments have led to the suggestion that Oncogene mediated repression of glucocorticoid hormone response ele- Rb engages at least two tumor suppressor functions that can be ments and glucocorticoid receptor levels. Cancer Res. 49:2266s–2274s. 17. Hu, N., A. Gutsmann, D. C. Herbert, A. Bradley, W.-H. Lee, and E. Y. Lee. genetically dissociated, one linked to E2F-dependent cell cycle 1994. Heterozygous Rb-1 delta 20/ϩ mice are predisposed to tumors of the progression and the other linked to control of Ras activation pituitary gland with a nearly complete penetrance. Oncogene 9:1021–1027. and differentiation (33, 58). 18. Ise, K., K. Kakamura, K. Nakao, S. Shimizu, H. Harada, T. Ichise, J. Miyoshi, Y. Gondo, T. Ishikawa, A. Aiba, and M. Katsuki. 2000. Targeted Our results indicate the existence of a novel, physiologically deletion of H-ras gene decreases tumor formation in mouse skin carcino- important genetic interaction between Rb and N-ras during genesis. Oncogene 19:2951–2956. 19. Jacks, T., A. Fazeli, E. M. Schmitt, R. T. Bronson, M. A. Goodell, and R. A. mammalian embryogenesis. N-ras participates in several of the Weinberg. 1992. Effects of an Rb mutation in the mouse. Nature 359:295–300. early and late developmental defects brought about by loss of 20. Jiang, Z., Z. Guo, F. A. Saad, J. Ellis, and E. Zacksenhaus. 2001. Retino- 5268 TAKAHASHI ET AL. MOL.CELL.BIOL.

blastoma gene promoter directs transgene expression exclusively to the ner- 47. Peeper, D. S., T. M. Upton, M. H. Ladha, E. Neuman, J. Zalvide, R. vous system. J. Biol. Chem. 276:593–600. Bernards, J. A. DeCaprio, and M. E. Ewen. 1997. Ras signalling linked to 21. Jiang, Z., P. Liang, R. Leng, Z. Guo, Y. Liu, X. Liu, S. Bubnic, A. Keating, the cell-cycle machinery by the retinoblastoma protein. Nature 386:177– D. Murray, P. Goss, and E. Zacksenhaus. 2000. E2F1 and p53 are dispens- 181. able, whereas p21Waf1/Cip1 cooperates with Rb to restrict endoreduplication 48. Perry, R. L. S., M. H. Parker, and M. A. Rudnicki. 2001. Activated MEK1 and apoptosis during skeletal myogenesis. Dev. Biol. 227:28–41. binds the nuclear MyoD transcriptional complex to repress transactivation. 22. Johnson, L., D. Greenbaum, K. Cichowski, K. Mercer, E. Murphy, E. Mol. Cell 8:291–301. Schmitt, R. T. Bronson, H. Umanoff, E. Windfried, R. Kucherlapati, and T. 49. Ramocki, M. B., S. E. Johnson, M. A. White, C. L. Ashendel, S. F. Konieczny, Jacks. 1997. K-ras is an essential gene in the mouse with partial functional and E. J. Taparowsky. 1997. Signaling through mitogen-activated protein overlap with N-ras. Genes Dev. 11:2468–2481. kinase and Rac/Rho does not duplicate the effects of activated Ras on 23. Jones, M. K., and J. H. Jackson. 1998. Ras-GRF activates Ha-Ras, but not skeletal myogenesis. Mol. Cell. Biol. 17:3547–3555. N-Ras or K-Ras 4B, protein in vivo. J. Biol. Chem. 273:1782–1787. 50. Ramocki, M. B., M. A. White, S. F. Konieczny, and E. J. Taparowsky. 1998. 24. Khosravi-Far, R., and C. J. Der. 1994. The Ras signal transduction pathway. A role for RalGDS and a novel Ras effector in the Ras-mediated inhibition Cancer Metastasis Rev. 13:67–89. of skeletal myogenesis. J. Biol. Chem. 273:17696–17701. 25. Koera, K., K. Nakamura, K. Nakao, J. Miyoshi, K. Toyoshima, T. Hatta, H. 51. Raptis, L., H. L. Brownell, M. J. Corbley, K. W. Wood, D. Wang, and T. Otani, A. Aiba, and M. Katsuki. 1997. K-Ras is essential for the development Haliotis. 1997. Cellular ras gene activity is required for full neoplastic trans- of the mouse embryo. Oncogene 15:1151–1159. formation by the large tumor antigen of SV40. Cell Growth Diff. 8:891–901.

26. Kong, Y., S. E. Johnson, E. J. Taparowsky, and S. F. Konieczny. 1995. Ras 52. Sasahara, R. M., C. Takahashi, and M. Noda. 1999. Involvement of the Sp1 Downloaded from p21Val inhibits myogenesis without altering the DNA binding or transcrip- site in ras-mediated downregulation of the RECK metastasis suppressor tional activities of the myogenic basic helix-loop-helix factors. Mol. Cell. gene. Biochem. Biophys. Res. Commun. 264:668–675. Biol. 15:5205–5213. 53. Sassoon, D., G. Lyons, W. E. Wright, V. Lin, A. Lassar, H. Weintraub, and 27. Kowenz-Leutz, E., G. Twamley, S. Ansieau, and A. Leutz. 1994. Novel mech- M. Buckingham. 1989. Expression of two myogenic regulatory factors myo- anism of C/EBP␤ (NF-M) transcriptional control: activation through dere- genin and MyoD1 during mouse embryogenesis. Nature 341:303–307. pression. Genes Dev. 8:2781–2791. 54. Schneider, J. W., W. Gu, L. Zhu, V. Mahdavi, and B. Nadal-Ginard. 1994. 28. Kratzke, R. A., G. A. Otterson, A. Hogg, A. B. Coxon, J. Geradts, J. K. Reversal of terminal differentiation mediated by p107 in RbϪ/Ϫ muscle cells. Cowell, and F. J. Kaye. 1994. Partial inactivation of RB product in a family Science 264:1467–1471. with incomplete penetrance of familial retinoblastoma and benign retinal 55. Screpanti, V. A., M. Maroder, E. Petrangeli, L. Frati, and A. Gulino. 1989. tumors. Oncogene 9:1321–1326. Tumor-promoting phorbol ester and ras oncogene expression inhibit the 29. Lamb, J., M. H. Ladha, C. McMahon, R. L. Sutherland, and M. E. Ewen. glucocorticoid-dependent transcription from the mouse mammary tumor 3: 2000. Regulation of the functional interaction between cyclin D1 and the virus long terminal repeat. Mol. Endocrinol. 1659–1665. http://mcb.asm.org/ estrogen receptor. Mol. Cell. Biol. 20:8667–8675. 56. Sears, R. C., and J. R. Nevins. 2002. Signaling networks that link cell pro- 30. Lasorella, A., M. Noseda, M. Beyna, and A. Iavarone. 2000. Id2 is a retino- liferation and cell fate. J. Biol. Chem. 277:11617–11620. blastoma protein target and mediates signalling by Myc oncoproteins. Na- 57. Sellers, W. R., and W. G. J. Kaelin. 1997. Role of the retinoblastoma protein ture 407:592–598. in the pathogenesis of human cancer. J. Clin. Oncol. 15:3301–3312. 31. Lee, E. Y.-H. P., C.-Y. Chang, N. Hu, Y.-C. J. Wang, C.-C. Lai, K. Herrup, 58. Sellers, W. R., B. G. Novitch, S. Miyake, A. Heith, G. A. Otterson, F. J. Kaye, W.-H. Lee, and A. Bradley. 1992. Mice deﬁcient for Rb are nonviable and A. B. Lassar, and W. G. J. Kaelin. 1998. Stable binding to E2F is not required show defects in neurogenesis and haematopoiesis. Nature 359:288–294. for the retinoblastoma protein to activate transcription, promote differenti- 32. Lee, E. Y.-H. P., N. Hu, S.-S. F. Yuan, L. A. Cox, A. Bradley, W.-H. Lee, and ation, and suppress tumor growth. Genes Dev. 12:96–106. K. Herrup. 1994. Dual roles of the retinoblastoma protein in cell cycle 59. Singh, P., J. Coe, and W. Hong. 1995. A role for the retinoblastoma protein regulation and neuron differentiation. Genes Dev. 8:2008–2021. in potentiating transcriptional activation of the glucocorticoid receptor. Na- 33. Lee, K. Y., M. H. Ladha, C. McMahon, and M. E. Ewen. 1999. The retino- ture 374:562–565. blastoma protein is linked to the activation of Ras. Mol. Cell. Biol. 19:7724– 60. Takahashi, C., N. Akiyama, T. Matsuzaki, S. Takai, H. Kitayama, and M.

7732. Noda. 1996. Characterization of a human MSX-2 cDNA and its fragment on October 13, 2019 by guest 34. Leone, G., J. DeGregori, R. Sears, L. Jakoi, and J. R. Nevins. 1997. Myc and isolated as a transformation suppressor gene against v-Ki-ras oncogene. Ras collaborate in inducing accumulation of active cyclin E/Cdk2 and E2F. Oncogene 12:2137–2146. Nature 387:422–426. 61. Thomas, D. M., S. A. Carty, D. M. Piscopo, J.-S. Lee, W.-F. Wang, W. C. 35. Lipinski, M. M., and T. Jacks. 1999. The retinoblastoma gene family in Forrester, and P. W. Hinds. 2001. The retinoblastoma protein acts as a differentiation and development. Oncogene 18:7873–7882. transcriptional coactivator required for osteogenic differentiation. Mol. Cell 36. Liu, Y., and E. Zacksenhaus. 2000. E2F1 mediates ectopic proliferation and 8:303–316. stage-specific p53-dependent apoptosis but not aberrant differentiation in 62. Tiemann, F., K. Masunuru, and P. W. Hinds. 1997. The retinoblastoma the ocular lens of Rb deficient fetuses. Oncogene 19:6065–6073. tumour suppressor protein and cancer, p. 163–185. In D. M. Swallow and 37. Lu, X., and H. R. Horvitz. 1998. lin-35 and lin-53, two genes that antagonize Y. H. Edwards (ed.), Protein dysfunction and human genetic disease. BIOS a C. elegans Ras pathway, encode proteins similar to Rb and its binding Scientific Publishers, Oxford, United Kingdom. protein RbAp48. Cell 95:981–991. 63. Trimarchi, J. M., and J. A. Lees. 2001. Sibling rivalry in the E2F family. Nat. 38. Lyons, G. E., S. Muhlebach, A. Moser, R. Masood, B. M. Paterson, M. E. Rev. Mol. Cell Biol. 3:11–20. Buckingham, and J. C. Perriard. 1991. Developmental regulation of creatine 64. Tsai, K. Y., Y. Hu, K. F. Macleod, D. Crowley, L. Yamasaki, and T. Jacks. kinase gene by myogenic factors in embryonic mouse and chick skeletal 1998. Mutation of E2f-1 suppresses apoptosis and inappropriate S phase muscle. Development 113:1017–1029. entry and extends survival of Rb-deficient mouse embryos. Mol. Cell 2:293–304. 39. Malumbres, M., and A. Pellicer. 1998. Ras pathways to cell cycle control and 65. Umanoff, H., W. Edelmann, A. Pellicer, and R. Kucherlapati. 1995. The cell transformation. Front. Biosci. 3:d887–d912. murine N-ras gene is not essential for growth and development. Proc. Natl. 40. Marshall, C. J. 1996. Ras effectors. Curr. Opin. Cell Biol. 8:197–204. Acad. Sci. USA 92:1709–1713. 41. Mittnacht, S., H. Paterson, M. F. Olson, and C. J. Marshall. 1997. Ras 66. Vooijs, M., and A. Berns. 1999. Developmental defects and tumor predis- signalling is required for inactivation of tumour suppressor pRb cell-cycle position in Rb mutant mice. Oncogene 18:5293–5303. control protein. Curr. Biol. 7:219–221. 67. Weinberg, R. 1993. Tumor suppressor genes. Neuron 11:191–196. 42. Nakajima, T., S. Kinoshita, T. Sasagawa, K. Sasaki, M. Naruto, and T. 68. Weinberg, R. A. 1995. The retinoblastoma protein and cell cycle control. Cell Kishimoto. 1993. Phosphorylation at threonine-235 by a ras-dependent mi- 81:323–330. togen-activated protein kinase cascade is essential for transcription factor 69. Wu, L., A. de Bruin, H. I. Saavedra, M. Starovic, A. Trimboli, Y. Yang, J. NF-IL6. Proc. Natl. Acad. Sci. USA 90:2207–2211. Opavska, P. Wilson, J. C. Thompson, M. C. Ostrowski, T. J. Rosol, L. A. 43. Novitch, B. G., G. J. Mulligan, T. Jacks, and A. B. Lassar. 1996. Skeletal Woollett, M. Weinstein, J. C. Cross, M. L. Rosinson, and G. Leone. 2003. muscle cells lacking the retinoblastoma protein display defects in muscle Extra-embryonic function of Rb is essential for embryonic development and gene expression and accumulate in S and G2 phases of the cell cycle. J. Cell viability. Nature 421:942–947. Biol. 135:441–456. 70. Yochem, J., M. Sundaram, and M. Han. 1997. Ras is required for a limited 44. Novitch, B. G., D. B. Spicer, P. S. Kim, W. L. Cheung, and A. B. Lassar. 1999. number of cell fates and not for general proliferation in Caenorhabditis pRb is required for MEF2-dependent gene expression as well as cell-cycle elegans. Mol. Cell. Biol. 17:2716–2722. arrest during skeletal muscle differentiation. Curr. Biol. 9:449–459. 71. Zacksenhaus, E., Z. Jiang, D. Chung, J. D. Marth, R. A. Phillips, and B. L. 45. Olson, E. N., G. Spitz, and M. A. Tainsky. 1987. The oncogenic forms of Gallie. 1996. pRb controls proliferation, differentiation, and death of skeletal N-ras and H-ras prevent skeletal myoblast differentiation. Mol. Cell. Biol. muscle cells and other lineages during embryogenesis. Genes Dev. 10:3051– 7:2104–2111. 3064. 46. Page, B. D., S. Guedes, D. Waring, and J. R. Pries. 2001. The C. elegans E2F- 72. Ziebold, U., T. Reza, A. Caron, and J. A. Lees. 2001. E2F3 contributes both and DP-related proteins are required for embryonic asymmetry and nega- to the inappropriate proliferation and to the apoptosis arising in Rb mutant tively regulate Ras/MAPK signaling. Mol. Cell 7:451–460. embryos. Genes Dev. 15:386–391.